In recent years, it turned out that metabolites in living organisms have been affected not only by genetic factors but by many environmental factors, and analysis of these metabolite has attracted attention as a guideline for diagnosis of diseases and for health condition, as well as for the analysis of genes and proteins. Among them, amino acid related compounds such as amino acids and amines are one of the metabolite groups existing in most large quantity (see, for example, Non-patent literature 1), the quantitative analyses thereof have been carried out for many years, and various methods of analysis have been developed.
On the other hand, in the quantitative analysis, usually, the calibration curve method, in which a calibration curve is made up by analyzing standard substances with multiple concentrations by interleaving a calibration point of the compound to be analyzed, is generally employed. In the quantitative analysis by such a calibration curve method, correlation between concentration and signal (specifically, a signal which indicates a peak area, peak height, etc.) is determined beforehand using external standard with known concentration, and the concentration in a specimen sample is determined based on the information (that is, the calibration curve) obtained thereupon. Therefore, in the quantitative analysis using the calibration curve method, the reference standard for quantitative determination will be quite important to achieve the analysis with high degree of accuracy since the calibration curve obtained from external standard (criteria) serves as a criteria. And, in order to carry out quantitative analysis by the calibration curve method with high degree of accuracy, it will be essential for performing highly accurate quantitative method that a series of several different concentrations of the reference standard is prepared, and a calibration curve is made up actually by using them, and the concentration of measuring object in a specimen sample fits within the concentrations of the reference standard which are used to make up the calibration curve. Specifically, if the concentration of the object in a specimen sample is 50 μM (μmol/L), it is preferable to use the standard substance of 25 μM to 75 μM. Furthermore, the range of calibration points (calibration points) of reference standard in this case, is preferable to be narrow since the linearity between concentration and signal can easily be secured. In more specifically, it is more desirable to take the calibration points from 25 μM to 75 μM rather than to take the calibration points from 1 μM to 100 μM.
However, the amino acids (amino acid related compounds) in the living organism include so many kinds. For example, in the inspection items of SRL Inc., 39 kinds of amino acid related compounds are listed as an object for analysis (see, for example, Non-patent literature 2). And, the amino acid concentration in the living organism varies greatly depending on the kind of amino acid, and it is known that there exists concentration difference in about 100 times (double digits) between amino acid with high concentration and amino acid with low concentration. Therefore, on the occasion of performing amino acid analysis using existing mixed standard solution [for example, Amino Acids Mixture Standard Solution, Type AN-II (code numbers: 015-14461, 011-14463), Type B (code numbers: 016-08641, 012-08643), Type H (code numbers: 013-08391, 019-08393), and so on (all are sold from Wako Pure Chemical Industries, Ltd.)], there were a problem of taking time for preparatory work such that depending on the concentration range of each amino acid related compound, dilution of the standard substance or specimen sample is needed.
On the other hand, although a report on the reference standard substance for the amino acids in human plasma has been published (Non-patent Reference 3), since the standard substance in said report was a mixed plasma pool of the plasma obtained from 100 men and women of unspecified number of Americans, the lot-to-lot variation of concentration value could not be avoided. In addition, in the case where the amino acid analysis is carried out using it as a standard solution, since the concentration of each kind of amino acid contained in this standard substance was the average of 100 persons, when the amino acid concentration in a specimen sample exceeds the average value, its concentration would deviate from the range of calibration curve, showing lacked reliability in measurement accuracy. Furthermore, since said standard substance was the one which includes various biological substances other than amino acid, it could not deny the possibility of influence of these substances on the measurement value, and had a problem of employing it as a standard substance for carrying out the quantitative analysis with high degree of accuracy.
Therefore, development of a standard substance that does not need to adjust its concentration depending on the kinds of amino acids to be measured, and by using a solution diluted appropriately from single solution, it can provide a calibration curve which enables to perform quantitative method of various kinds of amino acid in a sample derived from living organism in high accuracy has been desired.
Furthermore, in the quantitative analysis method employing the calibration curve method, there are so-called external standard method and internal standard method, etc., and regarding the internal standard substance, similarly to the case with the external standard substance, quantitative accuracy can be increased by designing the standard substance according to the nature of the specimen sample. In particular, when a mass spectrometer is used as a detector, since the impact of matrix effect in the sample is large, it will be very important to use the internal standards for carrying out measurement with a high degree of accuracy.
Although several researches have been reported on the measurement of amino acid employing this internal standard method (see, for example, Non-patent literature 4 and 5), conventional internal standard substances might sometimes show unfavorable linearity between concentration and signal, and had problems such that if these were used, accuracy of amino acid measurement would be not good. Therefore, development of an internal standard substance which solved such problem has been desired.